Systems and methods for performing microscopic analysis of a sample

ABSTRACT

The present description relates to a system for microscopic analysis of a sample that includes a microscopic analysis path and a sighting path. The microscopic analysis path notably includes an illumination path that illuminates the sample through a microscope objective of given nominal numerical aperture in a given field of view and a detection path including the microscope objective, and that detects in the field of view and according to a detection pattern, a light beam emitted by the sample in response to the illumination of the sample. The sighting path includes the microscope objective, a device for full field illumination of the sample, a two-dimensional detector, one or more imaging elements forming, with the microscope objective, a full field imaging device that forms a surface reflection sighting image of the sample of a given effective field encompassing the field of view.

TECHNICAL FIELD OF THE INVENTION

The present description relates to systems and methods for microscopicanalysis of a sample and relates in particular to the microscopicanalysis of a biological tissue, in particular skin.

PRIOR ART

In the context of a dermatological examination in particular, it isknown to carry out a dermoscopic examination, that is to say anobservation of the surface of the skin using a magnifying opticalinstrument, and then to carry out a local microscopic analysis accordingto the observations made on the wide-field image obtained by thedermoscopic examination.

The microscopic analysis comprises, for example, microscopic imaging orspectroscopic analysis.

Among the imaging techniques, there are known in particular, and in anon-limiting manner, confocal microscopy techniques such as, forexample, the technique described in Rajadhyaksha et al. [Ref. 1] or K.Konig et al. [Ref. 2] for nonlinear microscopy. Also known aretechniques of optical coherence tomographic microscopy (OCM), in thetime domain (time domain OCM) or in the frequency domain (frequencydomain OCM). The known OCM techniques include techniques that combineoptical coherence tomography and confocal microscopy (see, for example,Schmitt et al. [Ref. 3]) in order to improve lateral resolution.

More specifically, the patent application WO2015092019 [Ref. 4]describes a technique of visualizing the internal structure of asemi-transparent object arranged at the focus of a microscope objective,for example a biological tissue, in order to obtain vertical sections orB-scans orthogonal to the surface of the object, at a high rate (severalsections per second), with high spatial resolution, that is to say ofthe order of 1 μm, both axially and laterally, and a satisfactory depthof penetration, of the order of a millimeter. This technique is based onoptical coherence microscopy but has a confocal filtering configurationthat is linear or one-dimensional (in one direction); for this, theillumination line is optically conjugated, in particular by means of themicroscope objective, with a linear detector, the detection area ofwhich has a width substantially identical to a width of the image of theline, resulting in a spatial filtering of a region of the object that isto be observed. Such a technique is thus known as line-field confocaloptical coherence tomography (LC-OCT).

The article by Y. Chen et al. [Ref. 5] has also proposed a line-scanningoptical coherence tomographic microscopy device, but one in which asample is moved in a plane perpendicular to an optical axis of themicroscope objective, and in a direction perpendicular to theillumination line, making it possible to form en-face images of thesample (or C-scans).

Among the techniques for spectroscopic analysis of a sample, and inparticular of a biological tissue such as skin, there is known forexample, and in a non-limiting manner, Raman spectroscopy, which makesit possible to form a molecular fingerprint of biological tissues, asdescribed for example in Schleusener et al. [Ref. 6]. The review articleby E. Drakaki et al. [Ref. 7] generally presents different spectroscopytechniques applied to the microscopic analysis of skin.

All of the microscopic analysis techniques described above, whether forimaging or for spectroscopy, use a microscope objective having aconsiderable nominal numerical aperture, typically greater than or equalto 0.5, for a given field of view, typically of between about 0.2 mm andabout 1.5 mm.

In practice, in order to obtain relevant information during themicroscopic analysis, it is important for the practitioner to find,during the microscopic analysis, the zone that appears to him to besuspect in the image obtained during the dermoscopic examination.

However, precisely finding in microscopic analysis a suspect zone thathas been identified in dermoscopy is a complex matter, because theimages on which it is possible to rely for sighting are obtained on muchsmaller fields than in dermoscopy, and they have a very differentappearance. This is even more critical when the microscopic analysisdoes not produce images, such as in Raman microspectroscopy for example.

Different solutions have been proposed to allow a practitioner toidentify, on the dermoscopic image, the field of analysis for themicroscopic analysis.

The patent application WO2017139712 [Ref. 8] describes, for example, asystem which combines confocal microscopy (or reflectance confocalmicroscopy (RCM)) and wide-field dermoscopy (WFD). For this, amicro-camera is directly integrated in the microscope objective in orderto form a surface image in wide-field reflection. However, such a systemis complex to manufacture and to integrate; moreover, the imagesobtained by the micro-camera are of poor quality.

The U.S. Pat. No. 7,864,996 [Ref. 9] describes a confocal imaging systemcoupled with a dermatoscope. The dermatoscope is mounted on a modulefixed to the skin and makes it possible to image the same zone as theconfocal microscope, which can be fixed on the same module. Theacquisition of a dermoscopic image (or “macroscopic” image) isperformed, followed by the acquisition of confocal images. A precisecorrelation between the images is made in order to represent, on thedermoscopic image, the position of the image formed by the confocalimaging system. However, the system thus described requires anadditional module for fixing two separate probes, and it may bedifficult for this module to be fixed at any location on the skin. Inaddition, a complex procedure must be followed for the acquisition ofthe images in order to obtain the correlation of the dermoscopic andconfocal images.

In the case of Raman microspectroscopy, Z. Wu et al. [Ref 10] describehow to acquire and localize micro-Raman signals in tissues by means ofreflectance confocal microscopy imaging, and using a single lasersource. However, a confocal image is less easy to use, as a referenceimage for a practitioner, than a dermoscopic image.

The present description proposes microscopic analysis devices andmethods allowing a user to locate with precision, and by means of asimple acquisition method, the field of the microscopic analysis in awide-field surface reflection image, in which the image quality is closeto the quality of a dermoscopic image.

SUMMARY OF THE INVENTION

In the present description, the term “comprise” signifies the same thingas “include”, “contain”, and is inclusive or open and does not excludeother elements which are not described or shown. Moreover, in thepresent description, the term “about” or “substantially” is synonymouswith (signifies the same thing as) an upper and/or lower margin of 10%,for example 5%, of the respective value. According to a first aspect,the present description relates to a system for microscopic analysis ofa sample, comprising:

-   -   a microscopic analysis path comprising:        -   a microscope objective of given nominal numerical aperture            in a given field of view;        -   an illumination path configured to illuminate the sample            through the microscope objective according to a first            illumination pattern and in a first spectral band;        -   a detection path comprising said microscope objective, said            detection path being configured to detect in said field of            view, and according to a detection pattern, a light beam            emitted by the sample in response to said illumination of            the sample, and to generate a detection signal;        -   a processing unit configured to generate information on            microscopic analysis of the sample from said detection            signal;    -   a sighting path comprising:        -   said microscope objective;        -   a full-field illumination device configured to illuminate            the sample in a second spectral band;        -   a two-dimensional detector;        -   one or more imaging elements forming, with said microscope            objective, a full-field imaging device configured to            optically conjugate a given effective field of the sample            encompassing said field of view with a detection area of the            two-dimensional detector, and to form a sighting image in            surface reflection of said effective field;    -   a beam splitter element arranged upstream of the microscope        objective in order to separate the analysis path and the        sighting path;    -   a display module configured to show said sighting image and, on        said sighting image, an image element indicating the position of        said detection pattern.

In the present description, the term “field of view” of the microscopeobjective refers to a region of a focal plane of the microscopeobjective, located in the object space (sample space) for which themanufacturer guarantees a nominal numerical aperture. A nominalnumerical aperture is, for example, between about 0.1 and about 1.4, forexample between about 0.5 and about 0.9. The field of view can bedefined by a circle with a diameter of between about 100 μm and about 5mm, for example between about 500 μm and about 1.5 mm.

The term “effective field” of the microscope objective is a field in theobject space (sample space) which is included in a total field of themicroscope objective, which encompasses said field of view and whosedimensions are limited by the full-field imaging device of the sightingpath. The effective field can be defined by a circle with a diameter ofbetween about 1 mm and about 10 mm, for example between about 2 mm andabout 5 mm.

In the present description, the illumination pattern depends on theillumination path of the microscopic analysis path and can comprise anillumination point, an illumination line or an illumination surface, forexample a rectangular surface resulting from the scanning of anillumination point or of an illumination line. An illumination point ismore precisely defined as the diffraction pattern resulting from thefocusing, by the microscope objective of the microscopic analysis path,of a collimated light beam incident on said objective. The illuminationpattern can also comprise an illumination surface which does not resultfrom scanning, for example a surface with circular geometry, in the caseof a full-field microscopic analysis path. The light beam emitted by thesample in response to the illumination of the sample can be a reflectedbeam, a backscattered beam, or a beam resulting from an emission processat another wavelength (for example fluorescence, Raman scattering,etc.).

Moreover, the detection pattern is included in the field of view and isincluded in the illumination pattern or is of the same order ofmagnitude, and depends on the detection path of the microscopic analysispath. The detection pattern can comprise a detection point, a detectionline or a detection surface, for example a rectangular surface resultingfrom the scanning of a line, or, in the case of a full-field microscopicanalysis path, a surface optically conjugated with a detection area of adetector. A detection point is here defined in the object space by anelementary zone optically conjugated with an elementary detector of adetector of the detection path of the microscopic analysis channel.

The applicant has shown that the system for microscopic analysis of asample according to the first aspect allows a user to precisely locatethe field of the microscopic analysis in a wide-field surface reflectionimage or “sighting image”. Said wide-field surface reflection image canpresent an image quality close to the quality of a dermoscopic image dueto the fact that the sighting path is moved apart. However, the systemretains very good compactness compared to the systems of the prior artthat require two probes ([Ref 9] for example).

According to one or more exemplary embodiments, the full-field imagingdevice of the sighting path has, in the object space of the microscopeobjective, a numerical aperture strictly lower than the nominalnumerical aperture of the microscope objective. It is then possible forthe sighting path to benefit from an effective field greater than thefield of view while limiting aberrations and potential vignetting, whileat the same time maintaining limited dimensions for the imagingelement(s) forming the full-field imaging device. The quality of thesighting image is therefore further improved.

According to one or more exemplary embodiments, said sighting pathfurther comprises a diaphragm making it possible to limit the numericalaperture of the full-field imaging device. According to other exemplaryembodiments, it is directly one of said imaging elements forming thefull-field imaging device that is configured to additionally form adiaphragm for limiting the numerical aperture of the full-field imagingdevice. According to one or more exemplary embodiments, the full-fieldimaging device of said sighting path is adjustable in focusing. Thismakes it possible to form a sighting image in surface reflection of thesample even when the microscopic analysis path images deep into thesample (case of OCM imaging for example).

According to one or more exemplary embodiments, the full-fieldillumination device of the sighting path comprises a plurality of lightsources arranged on a periphery of a distal face of the microscopeobjective, that is to say the face of the microscope objective in thesample space. This configuration permits direct illumination of thesample. Alternatively, the full-field illumination device of thesighting path can comprise a source arranged upstream of the microscopeobjective and a beam splitter element, for example a splitter cube,configured to direct an illumination beam through the microscopeobjective, toward the sample.

According to one or more exemplary embodiments, the second spectral banddiffers at least partially from the first spectral band, and saidsighting path comprises means for reducing the light power at least insaid first spectral band. In some cases indeed, an illumination beam ofthe sample in the illumination path of the microscopic analysis path canhave a light power strong enough to dazzle the detector of the sightingpath. By reducing the light power at least in said first spectral band,such a risk of glare is limited.

According to one or more exemplary embodiments, the second spectral banddiffers at least partially from the first spectral band, and said beamsplitter element comprises a plate or a dichroic cube, configured toseparate the beams in each of said first and second spectral bands. Thedichroic plate then forms means for reducing the light power in saidfirst spectral band. According to one or more exemplary embodiments, themicroscopic analysis path comprises a device for scanning anillumination beam of the sample and a beam emitted by the sample inresponse to said illumination of the sample, and said beam splitterelement forms part of the scanning device.

According to one or more exemplary embodiments, said image elementindicating the position of said detection pattern comprises a graphicelement determined by means of a prior calibration. This configurationis particularly advantageous in particular when the illumination patternis not detected by the detector of the sighting path, for example eitherbecause the detector of the sighting path is not sensitive in the firstspectral band or because the first spectral band in the sighting path iscut in order to limit glare. This configuration is also advantageouswhen the illumination pattern is difficult to identify in the sightingimage, or if the detection pattern is substantially different from theillumination pattern.

According to one or more exemplary embodiments, said microscopicanalysis path is a confocal and/or optical coherence tomographic imagingpath, and said information on microscopic analysis of the samplecomprises at least one image of the sample. For example, the microscopicanalysis path is an optical coherence tomographic imaging path asdescribed in the prior art and is configured to form B-scans, C-scans(or en-face images) of the sample or 3D images of the sample. In knownmanner, a cross-sectional image of the sample, called a B-scan, is animage formed in a plane parallel to the optical axis of the microscopeobjective; a cross-sectional image of the sample called a C-scan, oren-face image, is an image formed in a plane perpendicular to theoptical axis of the microscope objective, and a 3D image of the sampleresults from the acquisition of a plurality of B-scan images or C-scansimages and thus permits an analysis of the sample in a volume.

According to one or more exemplary embodiments, said microscopicanalysis path is a spectroscopic analysis path, and said information onmicroscopic analysis of the sample comprises at least one spectrum ofsaid light beam emitted by the sample at at least one point of thesample. According to a second aspect, the present description relates toa method for analysis of a sample, comprising:

-   -   a microscopic analysis of the sample by means of a microscopic        analysis path comprising a microscope objective of given nominal        numerical aperture in a given field of view, said microscopic        analysis comprising:        -   illuminating the sample through the microscope objective            according to a first given illumination pattern and in a            first spectral band;        -   detecting in said field of view, and according to a            detection pattern, a light beam emitted by the sample in            response to said illumination of the sample in order to form            a detection signal;        -   processing said detection signal in order to generate            information on microscopic analysis of the sample;    -   the formation of a sighting image in surface reflection of a        given effective field of the sample encompassing said field of        view, by means of a sighting path comprising said microscope        objective, a two-dimensional detector, one or more imaging        elements configured to form with said microscope objective a        full-field imaging device, the formation of the sighting image        comprising:        -   full-field illumination of the sample in a second spectral            band;        -   optical conjugation of the effective field of the sample            with a detection area of the two-dimensional detector, by            means of said full-field imaging device, in order to form            said sighting image;    -   displaying said sighting image and, on said sighting image,        displaying an image element indicating the position of said        detection pattern.

According to one or more exemplary embodiments, the microscopic analysisof the sample and the formation of a sighting image are carried outcontinuously, which entails that the sources of illumination of theanalysis path and of the sighting path are both in operation when themicroscopic analysis system is in use. This configuration is possible inthe case in particular where an illumination beam of the sample in themicroscopic analysis path is invisible or very attenuated in thesighting path, or more generally when the illumination beam of thesample in the microscopic analysis path does not disturb the acquisitionof the sighting image.

According to one or more exemplary embodiments, the method for analysisof a sample according to the first aspect comprises:

-   -   a first step of forming a sighting image of the sample without        illumination of the microscopic analysis path,    -   the detection of an analysis zone of interest in the sighting        image of the sample, and    -   the microscopic analysis of said sample in said zone of        interest.

This configuration is interesting in particular in the case where anillumination beam of the sample in the microscopic analysis path candisturb the detection in the sighting path but the illumination of thesample in the sighting path does not disturb the detection in themicroscopic analysis path.

It is also possible to turn off the illumination of the sighting pathduring the microscopic analysis of the sample if the illumination of thesample in the sighting path disturbs the detection in the microscopicanalysis path. In this case, the microscopic analysis of the sample andthe formation of a sighting image are carried out successively.

According to one or more exemplary embodiments, the microscopic analysisof the sample comprises confocal and/or optical coherence tomographicimaging of the sample, making it possible to form B-scan, C-scan or 3Dimages of the sample.

According to one or more exemplary embodiments, the method furthercomprises the display of at least one of said B-scan and C-scan images,and/or, in the case of the formation of a 3D image, the display of atleast one of said B-scan and C-scan images extracted from the 3D image.

For example, the microscopic analysis of the sample comprises theformation of B-scan images with a given imaging rate, and said imagingrate is synchronized with a rate of acquisition of sighting images. Asthe acquisition of B-scan images may require scanning of theillumination beam deep in the sample, for example by means of an axialdisplacement of the microscope objective, the synchronization ensuresthat the sighting images are acquired with an identical position of themicroscope objective with respect to the surface of the sample.

According to one or more exemplary embodiments, the microscopic analysisof the sample comprises a spectroscopic analysis of the sample.

According to one or more exemplary embodiments, the method according tothe second aspect comprises a prior calibration step making it possibleto determine, for said image element, a graphic element indicating theposition of said detection pattern.

According to one or more exemplary embodiments, the method according tothe second aspect further comprises the display of a marker superimposedon said image element of the sighting image, said marker allowing a userto target a point of interest in the detection pattern. Thus, in certainembodiments, a user is able to position the marker on said image elementin order to obtain microscopic analysis information in the sample, atthe level of said marker. In certain exemplary embodiments, a user isalso able to select a point of interest at the level of the microscopicanalysis information and see the marker position itself at thecorresponding location of the image element. Thus, for example, in thecase where the microscopic analysis of the sample comprises theformation of B-scan and/or C-scan images of the sample, a user will beable to target a point in one of said images displayed simultaneouslywith the sighting image, for example by means of a reticle, and will beable to see the marker position itself on the sighting image, the markercorresponding to the projection of the targeted point on the surface ofthe sample. The user will also be able to position the marker on thetarget image and see a reticle position itself on one of said images, ina position corresponding to that of the marker.

According to one or more exemplary embodiments, the sample is abiological tissue, for example skin.

BRIEF DESCRIPTION OF THE FIGURES

Other advantages and features of the invention will become clear onreading the description, illustrated by the following figures:

FIG. 1A: a diagram illustrating a first example of a system formicroscopic analysis of a sample according to the present description;

FIG. 1B: a diagram illustrating a second example of a system formicroscopic analysis of a sample according to the present description;

FIG. 1C: a diagram illustrating a third example of a system formicroscopic analysis of a sample according to the present description;

FIG. 2 : a diagram illustrating an example of a sighting path of asystem for microscopic analysis of a sample according to the presentdescription;

FIG. 3A: a diagram illustrating a first example of a device forfull-field imaging of a sighting path of a microscopic analysis systemaccording to the present description;

FIG. 3B: a diagram illustrating a second example of a device forfull-field imaging of a sighting path of a microscopic analysis systemaccording to the present description;

FIG. 4 : a diagram illustrating different examples of a field of view,an effective field and a total field of the microscope objective, andalso different detection patterns;

FIG. 5A: diagrams illustrating calibration steps for determining, forsaid image element, a graphic element indicating the position of saiddetection pattern, according to an example applied to microscopicimaging;

FIG. 5B: diagrams illustrating calibration steps for determining, forsaid image element, a graphic element indicating the position of saiddetection pattern, according to an example applied to spectroscopicanalysis;

FIG. 6A: a first image illustrating an example of the display of asighting image and a microscopic image (B-scan) obtained by means of amethod according to the present description;

FIG. 6B: a second image illustrating the same example of the display ofa sighting image and a microscopic image (B-scan) obtained by means of amethod according to the present description;

FIG. 7 : images illustrating an example of the display of a sightingimage and microscopic images (B-scan and C-scan) obtained by means of amethod according to the present description.

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description, many specific details are setforth in order to provide a more in-depth understanding of the presentdescription. However, it will be apparent to a person skilled in the artthat the present description can be implemented without these specificdetails. In other cases, well-known features have not been described indetail, so as to avoid unnecessarily complicating the description.

Moreover, in order to ensure better clarity, the features are not shownto scale in the figures.

FIG. 1A shows a first system 101 for microscopic analysis of a sample S,in which the microscopic analysis path is a confocal imaging path withscanning (point or line), where the imaging may possibly be non-linearimaging. The microscopic analysis system 101 comprises a microscopeobjective 110 of given nominal numerical aperture NA in a given field ofview, a microscopic analysis path 140, which is a scanning confocalimaging path, and a sighting channel 150.

In this example, the microscopic analysis path 140 comprises anillumination path 120 configured to illuminate the sample through themicroscope objective 110 according to a given illumination pattern andin a first spectral band, and a detection path 130 comprising saidmicroscope objective 110, said detection path being configured to detectin the field of view, and according to a given detection pattern, alight beam emitted by the sample in response to said illumination of thesample. The microscopic analysis path 140 also comprises a processingunit 160 and a display module 170.

In this example, the illumination path 120 of the microscopic analysispath 140 comprises an illumination source 121 and a cylindrical lens ordeflection mirror 122 (optional). The illumination path also comprises asplitter element 141 (splitter cube or splitter plate) and a reflectingelement 142 (optional) which are configured to send an illuminationbeam, emitted by the illumination source 121, toward the microscopeobjective 110, and also a device 143 for scanning the illumination beam,configured to scan the illumination beam along one or two dimensions. Asplitter element 145 is configured to separate the sighting path 150 andthe microscopic analysis path 140. The splitter element 145 is, forexample, a splitter cube or a splitter plate having areflection/transmission ratio of between 10/90 and 90/10; it can beabout 50/50. Moreover, a platform 111 (optional) rigidly connected tothe microscope objective 110 permits an axial displacement 112 of theobjective with respect to the sample. The illumination source 121 cancomprise, for example, a source of emission of coherent (spatially),monochromatic and collimated light. Optics and/or spatial filters (notshown) can make the source collimated and/or coherent and/ormonochromatic. The wavelength of the source depends on the application.For confocal microscopy using reflection of the illumination beam offthe sample, and applied to imaging of the skin, a typical wavelength ofthe illumination source is about 800 nm. For confocal microscopy usingfluorescence or nonlinear microscopy, the wavelength can be adapted tothe wavelength of fluorescence excitation or of nonlinear emission ofthe sample. Depending on the applications, a polychromatic source canalso be used. Moreover, in nonlinear microscopy, for example in CARS orSRS microscopy, the source 121 can comprise a plurality of distinctemission sources (spatially coherent, monochromatic and collimated),which are combined via a cube or a plate. In the case offluorescence/nonlinear microscopy, a dichroic splitter element 141 willadvantageously be used which reflects the excitation wavelength andtransmits the emission wavelength of the sample (or vice versa). Thecylindrical optical element 122 is optional and permits microscopy withillumination along a line (so-called “line-field” microscopy).

The illumination beam scanner 143 can be configured for two-dimensionalscanning in order to form an image from the scanning of an illuminationpoint. In the case of a “line-field” system with a cylindrical lens ordeflection mirror 122, the illumination beam scanner 143 will be able tobe configured for one-dimensional scanning. The scanning device cancomprise one or more scanning elements chosen from among the followingelements: galvanometric mirrors, polygonal mirrors, electrical oracoustic-optical deflection systems, or a combination of these variouselements (in the case of bi-dimensional scanning). The scanning devicecan also include optics in order to conjugate at least one of saidscanning elements with an entrance pupil of the microscope objective110, for example in order to avoid vignetting.

In this example, the detection path 130 of the microscopic analysis path140 comprises a detector 138, the microscope objective 110, the scanningdevice 143, and the reflecting or partially reflecting elements 145, 142(optional), 141 configured to send a beam, emitted by the sample S inresponse to said illumination of the sample, toward the detector 138. Inthis example, the detection path 130 further comprises a lens 131configured to optically conjugate, with the microscope objective, aplane of the sample S with a detection area of the detector 138. Thelens 131 or “tube lens” can of course be composed of several opticallenses and can also be replaced by one or more reflecting elements, forexample a spherical or parabolic mirror.

The detector 138 comprises an optical sensor with a detection area andcan also include spatial filters for confocal detection, if this is notensured by the detection area dimensions, and/or spectral filters tolimit the wavelength band detected to the emission band of the sample inthe case of a fluorescence/nonlinear microscopy system. The sensor cancomprise an elementary detection surface (e.g. a photodiode) in the caseof a point scanning system, a one-dimensional sensor (e.g. a linearcamera) in the case of a “line-field” system, or a two-dimensionalsensor of which only a region of interest is considered in order toserve as an elementary detection area or one-dimensional sensor. It willbe noted that a two-dimensional sensor can also be used in a“conventional” way if a second scanning device similar to device 143 isplaced upstream of the sensor. The processing unit 160 receives, in aknown manner, a detection signal generated by the detector 138 andreconstructs microscopic images from the detection signal, for example a2D en-face image from a detection signal resulting from scanning of apoint or line illumination pattern, for example.

The processing unit is connected to a display module 170 configured torepresent a sighting image and, on the sighting image, an image elementindicating the position of the detection pattern, as will be illustratedin more detail below. The processing unit can also be connected to astorage unit (not shown) for storing the images and/or videos generated.

The microscopic analysis system 101 further comprises the sighting path150. As is illustrated in FIG. 1A, the sighting path 150 comprises themicroscope objective 110, the beam splitter 145, a full-fieldillumination device 158 configured to illuminate the sample in a secondspectral band, a two-dimensional detector 155 with a detection area 156,and one or more imaging elements represented in FIG. 1A by the elements151, 152 configured to form, with said microscope objective 110, afull-field imaging device which optically conjugates a given effectivefield of the sample with the detection area 156 of the two-dimensionaldetector 155. The sighting path thus makes it possible to form asighting image in surface reflection of the effective field which, aswill be described in more detail below, encompasses the field of view ofthe microscope objective.

In this example, the full-field illumination device 158 comprises aplurality of light sources which are arranged on a periphery of a distalface of the microscope objective 110 and allow direct illumination ofthe sample S. The light sources are, for example, light-emitting diodesemitting at wavelengths of between about 400 nm and about 800 nm. Ofcourse, other illumination devices are possible, for example a sourcearranged upstream of the microscope objective and a beam splitterelement, for example a splitter cube, configured to direct anillumination beam through the microscope objective, toward the sample.

As is shown in FIG. 1A, the two-dimensional detector 155 is connected,in this example, to the processing unit 160 for the acquisition anddisplay of the sighting image on the display module 170.

In operation, the sighting path 150 thus makes it possible to generate asighting image in surface reflection of the sample with a larger fieldthan the field of view of the microscope objective. Moreover, an imageelement which indicates the position of the detection pattern is shownon the sighting image, it being possible for the detection pattern to bea point, a line or a surface. It is thus possible for a user, forexample a practitioner, to precisely identify the field of themicroscopic analysis in the wide-field sighting image.

FIG. 1B shows a second example of a system 102 for microscopic analysisof a sample S, in which the microscopic analysis path is an opticalcoherence tomographic (OCT) microscopy path, for example a confocal OCTchannel, for example of the LC-OCT type as described, for example, in[Ref. 4] or [Ref. 5].

As in the preceding example, the microscopic analysis system 102comprises a microscope objective 110 of given nominal numerical aperture(NA) in a given field of view, the microscopic analysis path 140, whichis an optical coherence tomographic (OCT) path, the sighting channel150, a processing unit 160, and a display module 170. In this example,the sighting path 150 may be similar to the sighting path described withreference to FIG. 1A, only the microscopic analysis path 140 beingdifferent.

In the example of FIG. 1B, the microscopic analysis path 140 comprisesan illumination path 120 configured to illuminate the sample through themicroscope objective 110 according to a given illumination pattern. Inthis example, the illumination path comprises an illumination source121, a cylindrical lens or deflection mirror 122 (optional), a splitterelement 141 (splitter cube or splitter plate) and a reflecting element142 (optional) which are configured to send an illumination beam,emitted by the illumination source 121, toward the microscope objective110. In this example, the illumination path 120 also comprises a device143 for scanning the illuminating beam, configured to scan theillumination beam along one or two dimensions, a splitter element 145configured to separate the sighting channel 150 and the microscopicanalysis channel 140, and (optionally) a platform 111 rigidly connectedto the microscope objective 110 and (for example) to the splitterelement 141, configured for an axial displacement 112 of the objectivewith respect to the sample.

The illumination source 121 can comprise, for example, a source ofemission of coherent (spatially), polychromatic, collimated light.Optics and/or spatial filters (not shown) can make the source collimatedand/or coherent and/or with a specific spectral distribution. Thecentral wavelength of the source depends on the application, for exampleof between 600 nm and 1500 nm, and the spectral width for examplebetween 50 nm and about 250 nm. In the case of an LC-OCT application asdescribed for example in Ref. 4, the illumination source 121 cancomprise, for example, and in a non-limiting way, a supercontinuum laserspectrally filtered by an optical fiber for an emission of about 800 nmand collimated by an off-axis parabolic mirror. In the case of anapplication to full-field tomographic imaging or FF-OCT (full-fieldOCT), as described for example in the article by E. Beaurepaire et al.[Ref. 11], the illumination source 121 can be chosen to be spatiallynon-coherent and to comprise means for full-field illumination of thesample, for example a Kohler illumination system. The cylindricaloptical element 122 is optional and permits microscopy with illuminationalong a line (line-field microscopy).

The scanning device 143 for the illumination beam can be configured forone-dimensional or two-dimensional scanning of a point or a line inorder to form, in a known manner, a cross-sectional image of theso-called B-scan sample, that is to say in a plane parallel to theoptical axis of the microscope objective, a cross-sectional image of thesample called a C-scan, or en-face image, that is to say in a planeperpendicular to the optical axis of the microscope objective, or a 3Dimage of the sample resulting from the acquisition of a plurality ofB-scan images or C-scan images. As before, the scanning device cancomprise one or more scanning elements selected from among the followingelements: galvanometric mirrors, polygonal mirrors, electrical oracousto-optical deflection systems, or a combination of these differentelements (in the case of two-dimensional scanning). The scanning devicecan also include optics for conjugating at least one of said scanningelements with an entrance pupil of the microscope objective 110, forexample in order to avoid vignetting.

The detection path 130 of the microscopic analysis path is configured todetect a light beam emitted by the sample in response to saidillumination of the sample, according to a given detection pattern, butdiffers from the detection path of the microscopic analysis pathillustrated in FIG. 1A. In particular, the detection path comprises aninterferometer for implementation of optical coherence tomographicmicroscopy. More precisely, the interferometer comprises an object armwith the microscope objective 110, the scanning device 143 and thereflecting or partially reflecting elements 145, 142, 141, which areconfigured to send a beam, emitted by the sample S in response to saidillumination of the sample, toward a detector 138.

The interferometer of the detection path further comprises a referencearm, separated in this example from the object arm by the splitter cube141, and comprising in a known manner a microscope objective 133(optional), for example similar to the microscope objective 110 in orderto provide dispersion compensation, a dispersion compensation system(optional, not shown in FIG. 1B, especially in the case where there isno microscope objective 133), a reference mirror 135, a platform 134(optional) configured for example to cause the reference mirror 135 tomove when a modulation of the optical path on the reference arm isrequired. In this example, the detection path further comprises anobjective 131 configured to optically conjugate, with the microscopeobjective, a plane of the sample S with a detection area of the detector138.

As in the preceding example, the detector 138 comprises an opticalsensor with a detection area, and it can also include spatial filtersfor confocal detection, if this is not ensured by the dimensions of thedetection area, and/or spectral filters in order to limit the detectedwavelength band. The sensor can comprise an elementary detection surface(e.g. a photodiode) in the case of a point scanning system, aone-dimensional sensor (e.g. a linear camera) in the case of aline-field system, or a two-dimensional sensor of which only a region ofinterest is considered in order to serve as an elementary detection areaor as a one-dimensional sensor. In the case of an FF-OCT application, atwo-dimensional sensor can be used conventionally.

In operation, interferences are created at the detection area of thedetector 138 between the light coming from the reference arm and thelight backscattered by the sample illuminated according to theillumination pattern, optionally and in a known manner with a modulationof the path length difference between the reference arm and the objectarm of the sample, for the formation of tomographic images, inparticular en-face images. The processing unit 160 receives, in a knownmanner, detection signals generated by the detector 138 and resultingfrom the detection of interferences, and it is configured for thereconstitution of microscopic images from the detection signals, forexample images in 2D section (B-scan or C-scan). The processing unit 160is connected to a display module 170 configured to represent thesighting image and, on the sighting image, an image element indicatingthe position of the detection pattern, as will be illustrated in moredetail below. The processing unit can also be connected to a storageunit (not shown) for storing the images and/or videos generated.

Such a microscopic analysis path 140 thus functions as a known opticalcoherence tomographic microscopy channel from the prior art. Although aparticular example is shown in FIG. 1B, a person skilled in the art willunderstand that the microscopic analysis system according to the presentdescription applies to any assembly known from the prior art for opticalcoherence tomographic microscopy, and the optomechanical elements shownin FIG. 1B can be adapted accordingly.

According to exemplary embodiments, in the case of a microscopicanalysis path suitable for the formation of vertical cross-sectionalimages of the sample (B-scan) by scanning a line in depth, the formationof B-scan images will be able to be synchronized with a rate ofacquisition of the sighting images. Indeed, when the acquisition ofB-scan images comprises scanning of the illumination beam in depth inthe sample by means of a displacement of the microscope objective forexample, the synchronization makes it possible to ensure that thesighting images are acquired with an identical position of themicroscope objective with reference to the surface of the sample.

FIG. 1C shows a third example of a system 103 for microscopic analysisof a sample S, in which the microscopic analysis path is a spectroscopypath, for example Raman spectroscopy.

As in the preceding examples, the microscopic analysis system 103comprises a microscope objective 110 of given nominal numerical apertureNA in a given field of view, the microscopic analysis path 140, which isa spectroscopy path, and the sighting path 150.

In this example, sighting path 150 may be similar to the sighting pathdescribed with reference to FIG. 1A, only the microscopic analysis path140 being different. The microscopic analysis path 140 comprises anillumination path 120 comprising, in this example, an illuminationsource 121, a cylindrical lens or deflection mirror 122 (optional), asplitter element 141 (splitter cube or splitter plate) and a reflectingelement 142 (optional), which are configured to send an illuminationbeam, emitted by the illumination source 121, toward the microscopeobjective 110. In this example, the illumination path 120 also comprisesa scanning device 143 (optional) for the illumination beam, configuredto scan the illumination beam along one or two dimensions, a splitterelement 145 configured to separate the sighting path 150 and themicroscopic analysis path 140, and (optionally) a platform 111 rigidlyconnected to the microscope objective 110 and configured for axialdisplacement 112 of the objective with respect to the sample.

The illumination source 121 can comprise, for example, a source ofcoherent (spatially), monochromatic and collimated light. Apolychromatic source can also be used, for example in diffuse reflectionmicrospectroscopy. Optics and/or spatial filters (not shown) can makethe source collimated and/or coherent and/or monochromatic. Thewavelength of the source depends on the application. In Ramanmicrospectroscopy for example, applied to imaging of the skin, a typicalwavelength of the illumination source can be between about 780 nm andabout 830 nm.

The cylindrical optical element 122 is optional and permits microscopywith illumination along a line (line-field).

The detection path 130 of the microscopic analysis path is configured todetect a light beam emitted by the sample in response to saidillumination of the sample, according to a given detection pattern, butdiffers from the detection path of the microscopic analysis pathillustrated in FIG. 1A or in FIG. 1B. In particular, the detection pathcomprises, in addition to the microscope objective 110, the scanningdevice 143 (optional) and reflecting or partially reflecting elements145, 142, 141, a spectrometer. The spectrometer comprises, in thisexample and in a known manner, a spectral dispersion element 132, forexample a grating, an objective 133, and a detector 134, comprising asensor with a one-dimensional or two-dimensional detection area. Atwo-dimensional sensor makes it possible, in a line-field configuration,to measure a spectrum for each point of the detection pattern(measurement of several spectra in parallel, each line of the 2D sensorthus corresponding to the spectrum of a point in the detection pattern).The processing unit 160 receives, in a known manner, detection signalsgenerated by the detector 134 of the spectrometer for the reconstructionof spectroscopic signals at one or more points of the sample. Theprocessing unit 160 is connected to a display module 170 configured torepresent the sighting image and, on the sighting image, an imageelement indicating the position of the detection pattern, as will beillustrated in more detail below. The processing unit can also beconnected to a storage unit (not shown) for storing the images and/orvideos generated.

Such a microscopic analysis path 140 therefore functions as aspectroscopy path known from the prior art. Although a particularexample is shown in FIG. 1C, a person skilled in the art will understandthat the microscopic analysis system according to the presentdescription applies to any assembly known from the prior art forspectroscopy, and in particular Raman spectroscopy, and theoptomechanical elements represented in FIG. 1C can be adaptedaccordingly.

As before, in operation, the sighting path 150 makes it possible togenerate a sighting image in surface reflection of the sample S with alarger field than the field of view of the microscope objective.Moreover, an image element which indicates the position of the detectionpattern of the microscopic analysis path is represented on the sightingimage, it being possible for the detection pattern to be a point, a lineor a surface. It is thus possible for a user, for example apractitioner, to precisely identify the field of the microscopicanalysis in the wide-field sighting image. In each of the examplesillustrated in FIGS. 1A, 1B or 1C, the splitter element 145 configuredto separate the sighting path 150 and the microscopic analysis path 140is arranged to transmit a beam, emitted by the sample in response tosaid illumination of the sample by the illumination device 158 of thesighting path, toward the two-dimensional detector 155 and to reflect abeam, emitted by the sample in response to said illumination of thesample by the illumination path 120 of the microscopic analysis path,toward the detection path 140. Of course, it would be possible to makethe splitter element 145 work in reflection on the sighting path and intransmission on the microscopic analysis path. Moreover, when themicroscopic analysis path comprises a scanning device (143, FIGS.1A-1C), the beam splitter element may form part of the scanning device.

Whether in one or other of the configurations, the splitter element canbe configured to limit the light power in the sighting path 150 of thelight coming from the illumination path 120 of the microscopic analysispath and reflected by the sample. Indeed, the light, for example comingfrom a laser source, can be powerful and likely to cause glare in thesighting path. Thus, it is possible to use a splitter element with areflection coefficient different from the transmission coefficient (forexample a glass slide). To reduce the light power in the sighting path,it is also possible to add an optical density in the sighting path 150(downstream of the splitter element 145).

In the case where the spectral band of the illumination source 121 ofthe illumination path 120 of the microscopic analysis path is at leastpartially different from the spectral band of the illumination device158 of the sighting path, it will be possible for the splitter elementto further comprise a dichroic element, for example a plate or adichroic cube.

It is also possible to provide means for reducing the light power in thesighting path, these means for reducing the light power possiblycontaining a spectral filtering element when the spectral band of theillumination source 121 of the illumination path 120 of the microscopicanalysis path is at least partially different from the spectral band ofthe illumination device 158 of the sighting path.

It is also possible not to activate the illumination of the sighting andmicroscopic analysis paths continuously, in the event that illuminationof one of the paths could interfere with detection on the other path.

Thus, in practice, the microscopic analysis of the sample and theformation of a sighting image can be carried out continuously. This isthe case when the image element is directly the image, formed by thewide-field imaging device of the sighting path, of the illuminationpattern of the microscopic analysis path. This may also be the case whenthe image element is a graphic element indicating the position of thedetection pattern and when the illumination of the microscopic analysispath does not interfere with the detection of the sighting path, forexample because it is greatly attenuated in the sighting path, andreciprocally.

In other exemplary embodiments, the method can comprise a first step offormation of a sighting image of the sample with the illumination of themicroscopic analysis path turned off, the detection of an analysis zoneof interest in the sighting image of the sample, then the microscopicanalysis of the sample in said zone of interest, for example by movingthe sample in order to bring the graphic element, previously calibratedto indicate the detection zone, to the level of the analysis zone ofinterest.

This configuration is of interest in the case where the illumination ofthe microscopic analysis path may interfere with detection in thesighting path.

In some cases, the sighting channel can operate continuously, both forillumination and for acquisition, if the illumination of the sightingpath does not interfere with the detection of the microscopic analysispath. This makes it possible to have a continuous sighting image, evenif it is degraded during the time when the illumination of themicroscopic analysis path is activated.

In other cases, the illumination of the sighting path can be turned offduring the microscopic analysis of the sample, for example when theillumination of one path interferes with the detection on the other pathand it is not possible to simultaneously maintain the illumination onboth paths in order to obtain usable results.

FIG. 2 shows a diagram which illustrates an example of a sighting pathof a system for microscopic analysis of a sample according to thepresent description. The sighting path shown in FIG. 2 is in particularconfigured to operate with any of the systems shown as examples in FIGS.1A, 1B or 1C. In FIG. 2 , only the detection part of the sighting pathis shown, the illumination possibly comprising, as illustrated in FIGS.1A to 1C, a set of light sources arranged on the distal part of themicroscope objective, or any other device for full-field illumination ofthe sample S.

As is illustrated in FIG. 2 , the sighting path comprises the microscopeobjective 110, of which the exit pupil is indicated by reference sign115, and a two-dimensional detector, represented in FIG. 2 by adetection area 156, and an objective 253.

In this example, the sighting path further comprises a tube lens 251 andan eyepiece 252. These imaging elements form, together with theobjective 253 and the microscope objective 110, a full-field imagingdevice 250 configured to optically conjugate a given effective field ofthe sample encompassing said field of view with the area of detection156 of the two-dimensional detector.

Thus, unlike certain systems known from the prior art and in particular[Ref. 8], which describes a micro-camera integrated in the object spaceof the microscope objective, the sighting path according to the presentdescription, by virtue of being moved apart from the object space, makesit possible to form a sighting image in surface reflection of a field ofthe sample, called the effective field in the present description, whichincludes the field of view of the microscope objective, the sightingimage being able to have a very good optical quality without affectingthe object space of the objective. The dimensions of the effective fieldare limited by the full-field imaging device of the sighting path. Theeffective field can be defined by a circle with a diameter of betweenabout 2 mm and about 5 mm.

To further improve the optical quality of the sighting image, it isadvantageous for the full-field imaging device to have a numericalaperture, measured in the object space of the microscope objective,strictly lower than the nominal numerical aperture of the microscopeobjective.

Indeed, in a conventional microscopic analysis path, it is known to usea microscope objective with a high numerical aperture (NA), for examplean NA of between about 0.5 and about 1.25. This numerical aperture isguaranteed by the manufacturer for a nominal field, called the field ofview in the present description, which can be between about 500 μm andabout 1.5 mm.

However, because the microscope objective 110 is not used in thesighting path under nominal conditions of use, the resolution accessibleat the level of the sighting path may differ from the one announced inthe specifications of the objective. In particular, by using themicroscope objective with an effective field greater than the nominalfield of view, aberrations and/or vignetting may adversely affect thequality of the image. In order to obtain a better image quality for thesighting path, it is therefore possible to design a full-field imagingdevice which has a numerical aperture strictly lower than the nominalnumerical aperture of the microscope objective, for example a numericalaperture of between about 0.05 and 0.1.

An originality of the microscopic analysis system according to thepresent description is thus to be able to use the same microscopeobjective in two different optical paths, with possibly differentnumerical apertures: in the microscopic analysis path in which themicroscope objective is used under nominal conditions (high numericalaperture, high resolution and low field), and in the sighting path inwhich the microscope objective is combined with other optical elementsto form a full-field imaging device which optionally has a lowernumerical aperture, for example about 0.08, a lower resolution and awide field. The microscopic analysis system according to the presentdescription can thus be seen as two microscopes operating in parallelvia a single microscope objective.

In practice, the numerical aperture of the full-field imaging device 250of the sighting path can be limited by placing a diaphragm 255 in thesighting path, for example in a plane substantially conjugate with aplane of the exit pupil 115 of the microscope objective. FIGS. 3A and 3Bthus illustrate two examples for the reduction of the numerical apertureof the full-field imaging device 250. In these examples, the microscopeobjective 110 is not shown.

FIG. 3A shows a diagram illustrating a first example in which thesighting path comprises a diaphragm 255 making it possible to limit thenumerical aperture of the full-field imaging device.

FIG. 3B shows a diagram illustrating a second example in which thenumerical aperture of the full-field imaging device 250 of the sightingpath is limited by one of the optical elements of the full-field imagingdevice, in this example the objective 253 of the camera. Thisconfiguration is particularly of interest because it makes it possibleto reduce the size of the full-field imaging device 250 and thus toobtain a very compact sighting path. Of course, the numerical apertureof the full-field imaging device 250 of the sighting path could belimited by another of the optical elements of the full-field imagingdevice, for example the eyepiece 252. It will be noted that it is notessential for the diaphragm (255 in FIG. 3A or 253 in FIG. 3B) to beperfectly conjugate with the plane of the pupil of the microscopeobjective. However, if the plane of the diaphragm is substantiallyconjugate with the plane of the exit pupil of the microscope objective(as is shown for example in FIG. 2 ), this makes it possible not to losethe property of telecentricity of the microscope objective and toprevent rays from being vignetted within the microscope objective 110.

Moreover, the full-field imaging device 250 illustrates an example ofthe design of the sighting path, but other examples are possible. Forexample, the full-field imaging device 250 of the sighting path mightnot comprise an eyepiece 252, the camera objective 253 imaging a finitedistance, or the eyepiece 252 might not return the rays to infinity. Inany case, as has been explained above, it is advantageous to reduce thenumerical aperture (NA) of the device 250, in the object space of themicroscope objective, compared to the nominal NA of the microscopeobjective, whether by means of a diaphragm added in the sighting path orby means of one of the optical elements of the sighting path configuredto form a diaphragm.

Moreover, the full-field imaging device of the sighting path can also beadjustable in focus. This makes it possible to form a sighting image insurface reflection of the sample even when the microscopic analysis pathis configured to form an image deep in the sample (case of OCM imagingas illustrated in FIG. 1B for example).

Indeed, when the microscopic analysis path is an LC-OCT path forexample, the microscope objective 110 is caused to be translatedvertically, that is to say along its optical axis. The translation canbe dynamic in a mode of vertical scanning of the illumination line(obtaining B-scans), that is to say in a direction parallel to theoptical axis of the microscope objective, or controlled by the user in amode of horizontal scanning of the illumination line, that is to say ina direction contained in a plane perpendicular to the optical axis ofthe microscope objective (obtaining C-scans). In the sighting path,however, it is desired that the microscope objective continues to imagethe surface of the sample, for example the surface of the skin, whichremains at the same position when the microscope objective istranslated. In order to maintain optimal image quality for the sightingimage, it may therefore be useful to be able to modify the focusing ofthe wide-field imaging device 250 of the sighting path in order tomaintain an optical conjugation between the surface of the sample S andthe detection area 156 (FIG. 2 ).

To do this, it is possible to provide for one of the optical elements,for example the objective 253, a lens with variable focal length, or toprovide that this objective can be moved, for example using apiezoelectric motor, the detection area being held fixed. In practice,the adjustment of the focusing can be automatic (autofocus), which makesit possible to limit the adjustments that have to be made by the user.However, when the sample is the skin for example, there are not alwaysenough clear structures to allow the autofocus to be performed in aneffective way. Another possibility is then to calibrate the adjustablefocusing of the wide-field imaging device 250 of the sighting path insuch a way as to associate the correct focusing position with eachposition of the microscope objective within its travel.

FIG. 4 shows, by way of illustration, various examples showing the fieldof view 401 of the microscope objective 110, for which a nominalnumerical aperture is guaranteed by the manufacturer, the effectivefield 402 of the sighting path, and a total field 403 of the microscopeobjective. The total field of the microscope objective is a region of afocal plane of the microscope objective comprising all the points fromwhich a light ray can be collected by the objective. In practice, theeffective field 402 is chosen to be large enough to obtain a wide-fieldsighting image, but smaller than the total field in order to keep asighting image of sufficient optical quality.

On these three examples are shown three detection patterns of themicroscopic analysis path, namely a line 431, a surface 432, and a point433. In all cases, the detection pattern is included in the field ofview of the microscope objective.

A method for microscopic analysis according to the present descriptioncan be implemented by means of a microscopic analysis system asdescribed, for example and in a nonlimiting manner by means of one ofthe systems 101, 102, 103 described with reference to FIGS. 1A, 1B and1C respectively.

The method comprises the microscopic analysis of the sample S, forexample a biological tissue such as skin, by means of a microscopicanalysis path 140 as described for example with reference to FIGS. 1A,1B or 1C, and the formation of a sighting image of the sample by meansof a sighting path 150 as described for example with reference to FIGS.1A-1C and FIGS. 2 and 3A-3B above. The sighting image is a reflectionimage of an effective field 402 (FIG. 4 ) of the sample comprising thefield of view 401, as has been explained above.

The method for microscopic analysis according to the present descriptionfurther comprises the display, on the sighting image, of an imageelement indicating the position of the detection pattern (for example adetection pattern 431, 432 or 433 as shown in FIG. 4 ).

In exemplary embodiments of a method according to the presentdescription, the image element can be directly the image formed, by thewide-field imaging device of the sighting path, of the illuminationpattern formed on the sample by the illumination path 120 of themicroscopic analysis path 140 (see FIGS. 1A, 1B and 1C).

However, in certain exemplary embodiments, the image element can be agraphic element indicating the position of the detection pattern anddetermined by means of a prior calibration step.

This configuration is particularly advantageous especially when theillumination pattern is not detected by the detector of the sightingpath, for example either because the detector of the sighting path isnot sensitive in the spectral band of the illumination source of themicroscopic analysis path, or because the first spectral band is cut inthe sighting path in order to limit glare. This configuration is alsoadvantageous when the illumination pattern is difficult to identify inthe sighting image, or if the detection pattern is substantiallydifferent from the illumination pattern. FIG. 5A thus shows diagramsillustrating, according to a first exemplary embodiment applied tomicroscopic imaging, calibration steps for determining, for said imageelement, a graphic element indicating the position of the detectionpattern of the microscopic imaging path, in this example a detectionpattern formed of a rectangular surface. The calibration method isimplemented with a calibration sample which has a sharp edge, forexample the edge of a glass slide cut “at right angles”, or the edge ofa pattern printed by photolithography on a glass slide.

A first step 501 involves acquisition of a sighting image 510 of thecalibration sample and a microscopic en-face image 520 such that a sharpedge of the sample visible on the sighting image is visible on an edgeof the microscopic image 520, in this example a right edge. The line 531of the sighting image is then recorded as being the right edge of thedetection area of the microscopic analysis path. The method is repeatedin a second step 502 by moving the sample so that this time the sharpedge of the sample is situated on another side of the microscopic image,in this example the left edge. In the same way, the image 532 of thesighting image is recorded as being the left edge of the detection areaof the microscopic analysis path. The method is repeated in steps 503,504 in the same way, each time moving the sample in order to make thesharp edge appear on a new side of the microscopic image 520. Acorresponding line 534, 535 is recorded each time on the sighting image.As is illustrated in diagram 505, starting from the 4 lines recorded onthe sighting image, it is possible to reconstruct a graphic elementwhich indicates the position of the detection pattern, in this example arectangular surface which can be materialized by a rectangle on thesighting image during the acquisition of a microscopic image. Thecalibration thus makes it possible to perfectly identify the detectionpattern of the microscopic analysis path in the sighting path, and thisindependently of the sample that is analyzed. The calibration can beadapted to a line or point detection pattern.

FIG. 5B shows diagrams illustrating, according to a second exampleapplied to spectroscopic analysis, for example Raman spectroscopicanalysis, calibration steps for determining, for said image element, agraphic element indicating the position of the detection pattern, inthis example a detection point.

In a first step 541, a sighting image 510 of the calibration sample isacquired, and a Raman signal (561) is measured at the same time.

The reference sample is moved, for example from left to right, until astrong Raman signal (562) is observed. This corresponds to a first sharpedge of the calibration sample 551 that is recorded. The method isrepeated in a second step 542 by moving the calibration sample, forexample from bottom to top, until again a strong Raman signal appears(spectrum 562). This corresponds to a second sharp edge of thecalibration sample 552 that is recorded. As is illustrated in step 543,starting from the two straight lines 551, 552 recorded, it is possibleto determine a graphic element representative of the detection pattern530 (here a disk centered on the detection point) and positioned at theintersection of the two straight lines. It is possible to ensure theprecision of the calibration by repeating the steps 541, 542 but bygoing, for example, from right to left and then from top to bottom. Thecalibration thus makes it possible to perfectly identify the detectionpattern of the microscopic analysis path in the sighting path, and thisindependently of the sample analyzed.

In practice, a method for the microscopic analysis of a sample, forexample the analysis of the skin of a patient, can be carried out in thefollowing way by a practitioner, for example a dermatologist, byimplementing the steps of a method according to the present description.

In a first step, a visual examination of the skin is carried out.Clinical images (photos) can be taken in order to locate “suspect”structures on a body scale. A dermoscopic examination follows. Thedermatologist takes images of the suspect structure using a magnifyingoptical system, for example a dermatoscope, which optically correspondsto a magnifying glass, either digital or non-digital, with integratedillumination. The field of view of the dermatoscope is typically 1 to 3cm. Dermoscopic images can be recorded directly with a digitaldermatoscope or with the aid of a camera.

If any doubt persists during the dermoscopic examination, thedermatologist proceeds to a microscopic analysis of the skin, forexample by means of a system as illustrated in FIG. 1B, for example witha microscopic analysis path of the LC-OCT type. The dermatologistpositions the manual probe head (that is to say the part of the systemin contact with the skin for imaging) as close as possible to thesuspect structure. He then moves the whole probe (while keeping it incontact with the skin) until he precisely locates the lesion previouslyidentified in dermoscopy, being guided by the sighting path.

Once the structure is identified on the sighting image, thedermatologist proceeds to analyze the skin at the cellular level and indepth by virtue of the LC-OCT microscopic analysis path.

The examination begins, for example, with the vertical section imagingmode (B-scan), which gives access directly to the entire depth of thestructure. By virtue of the image element displayed on the macro imageindicating the position of the detection pattern (a line in the case ofthe cross-sectional imaging mode), the practitioner knows perfectly atwhat level in the structure he is in the process of observing a verticalsection at the cellular scale.

The dermatologist may also be interested in the LC-OCT image in order tosearch for pathological markers at the cellular level/deep within theskin, in order to enrich the information already obtained by dermoscopy.At this stage, it is possible to move around in the structure in orderto look for these pathological markers or to study them.

This movement can be done in two ways. Laterally by virtue of thescanning device (143 in FIG. 1B) present in the device, which makes itpossible to scan the detection pattern. The amplitude of this scan isquite low (˜500 μm), and in only one direction. The second way is tomove the whole probe or the skin under the probe. This displacement willa priori be less fine (its precision will depend on the control that theuser is able to exercise), but the dermatologist will have as muchamplitude as he wishes to target any zone in LC-OCT. For this type ofmovement, the sighting image is important because it allows thedermatologist to ensure that he always remains at the level of thestructure during his action in moving the probe or the skin.

Once the markers have been identified by LC-OCT, several options arepossible. The dermatologist can switch to horizontal section mode(C-scan or en-face) in order to enrich his understanding of thestructure (with the same approach to navigating the structure as invertical section mode).

It is also possible to acquire information within a volume of the samplein order to study pathological markers in 3D in a zone of interest.Following the acquisition of one or more volumes, the dermatologist canstop the acquisition system and study the volumes acquired for analysis.It should be noted that the recording of a volume is accompanied by therecording of a certain number of sighting images acquired during the 3Dacquisition (similarly, the recording of any image/video is accompaniedby the recording of the associated sighting image/video). Severalsighting images are recorded during a 3D acquisition in the case wherethe practitioner has moved during the acquisition (3D acquisitions canbe relatively long).

FIGS. 6A, 6B and 7 show images illustrating examples of sighting imagesand microscopic B-scan and C-scan images recorded during an acquisitionof a volume by means of a method according to the present description.In these examples, the microscopic analysis path is an LC-OCT typeanalysis path, as shown for example in FIG. 1B, configured foracquisition of 3D images. To obtain these images, the source used (121in FIG. 1B) is a supercontinuum laser filtered by an optical fiber foran emission at about 800 nm and collimated by an off-axis parabolicmirror. The microscope objective 110 is an objective immersed insilicone oil with 20× magnification and a numerical aperture NA=0.5. Acylindrical lens 122 with a focal length of 50 mm permits illuminationalong a line of ˜1.5 mm×1.5 μm at skin level, with a power of −10 mW. Agalvanometric scanner (143 in FIG. 1B) is used for lateral scanning ofthe illumination line. The fold mirror 142 is mounted on a piezoelectricactuator for the modulation of the interferences for the generation ofthe C-scan images. A microscope objective 133 identical to objective 110is used in the reference arm for dispersion compensation. The referencesurface 135 of the interferometer is an air/glass interface of a glassslide made of fused silica. The tube lens 131 has a focal length of 150mm, and detector 138 comprises a CMOS line-scan camera. A 90:10 splitterplate (145) is mounted on the scanning device 143 in order to separatethe microscope and sighting paths. The sighting path 150 comprises acamera 155 equipped with miniature optics of a few mm, serving as adiaphragm, as described in FIG. 3B. The camera is equipped withfocus/autofocus adjustment.

FIGS. 6A and 6B thus illustrate, by way of example, a B-scan image 620displayed with a sighting image 610. On the sighting image 610, thegraphic element 630 indicates the position of the detection pattern(line) of the microscopic analysis path. The graphic element isdetermined by virtue of a prior calibration, as described for examplewith reference to FIG. 5A.

A marker 640 can further be superimposed on the sighting image 610 inorder to mark a point on the graphic element, or more generally on theimage element, so as to allow a user to target a point in the B-scanimage 620 and to visualize the position of the point thus targeted atthe level of the surface of the sample on the sighting image. Forexample, the targeted point in the image 620 is indicated by a reticle641. The marker is calibrated to position itself at the levelcorresponding to the position of the targeted point via the reticle,projected onto the surface of the sample. Similarly, it is possible toleave the possibility to the user of targeting a point in the sightingimage, within the detection pattern, directly via the marker 640, so asto visualize to which position corresponds a point of the detectionpattern within the B-scan image (marked in this case by the verticalaxis of the reticle).

FIG. 6B illustrates the same image with movement of the marker 640 andof the reticle 641, the user being able either to move the reticle 641so as to see the marker reposition itself on the sighting image 610, orto move the marker 640 so as to see the reticle reposition itself on theB-Scan image 620.

FIG. 7 shows two images extracted from a volume, namely a B-scan image721 and a C-scan image 722. The sighting image 710 is displayed at thesame time with a detection pattern 730 corresponding to a detectionsurface of the C-scan.

Just as in the case of the B-scan image (FIG. 6A and FIG. 6B), a marker740 can be superimposed on the graphic element of the sighting image, ormore generally on the image element. The marker is calibrated so as tobe able to associate a point of the volume, marked here by a reticle741, with a point of the detection pattern marked by the marker, at thelevel of the surface of the sample.

The user can then target a point via the marker 740, respectively viathe reticle 741, so as to visualize to which position corresponds apoint of the volume projected onto the surface of the sample, within thedetection pattern, respectively to which position corresponds a point ofthe detection pattern within the volume, marked in this case by the axesof the reticle that can be viewed in the C-scan images extracted fromthe volume.

As is illustrated in these images, the method according to the presentdescription allows the practitioner to precisely identify the field ofthe microscopic analysis (in this example B-scans and C-scans) in thesighting image, which has an image quality close to the quality of adermoscopic image.

Although described through a number of exemplary embodiments, the methodand the system for microscopic analysis according to the presentdescription include variants, modifications and improvements which willbe obvious to a person skilled in the art, it being understood thatthese variants, modifications and improvements form part of the scope ofthe invention as defined by the claims that follow.

REFERENCES

-   Ref. 1: M. Rajadhyaksha et al, “In vivo confocal scanning laser    microscopy of human skin II: Advances in instrumentation and    comparison with histology”, J Invest Dermatol, 1999.-   Ref 2: K. Konig et al, “High-resolution multiphoton tomography of    human skin with subcellular spatial resolution and picosecond time    resolution,” J. Biomed. Opt. 8, 432-439 (2003).-   Ref. 3: Schmitt et al., “Subsurface Imaging of Living Skin with    Optical Coherence Microscopy”, Dermatology 1995; 191:93-98.-   Ref 4: Published patent application WO2015092019.-   Ref. 5: Y. Chen et al. “High-resolution line-scanning optical    coherence microscopy”, Optics Letters, vol. 32, no. 14, 1971-1973    (2007).-   Ref. 6: J. Schleusener et al., “Raman spectroscopy for the    discrimination of cancerous and normal skin”, Photon Lasers Med    (2015).-   Ref 7: E. Drakaki et al. “Spectroscopic methods for the    photodiagnosis of nonmelanoma skin cancer”, Journal of Biomedical    Optics 18(6), 061221 (June 2013).-   Ref. 8: Published patent application WO2017139712.-   Ref. 9: Granted patent U.S. Pat. No. 7,864,996.-   Ref 10: Z. Wu et al. “Precise in vivo tissue micro-Raman    spectroscopy with simultaneous reflectance confocal microscopy    monitoring using a single laser”, vol. 44, no. 6/15 Mar. 2019/Optics    Letters.-   Ref. 11: E. Beaurepaire et al. “Full field optical coherence    microscopy” Opt. Lett. 23, 244-246 (1998)

1. A system for microscopic analysis of a sample (S), comprising: amicroscopic analysis path comprising: a microscope objective of givennominal numerical aperture (NA) in a given field of view; anillumination path configured to illuminate the sample through themicroscope objective according to a first illumination pattern and in afirst spectral band; a detection path comprising said microscopeobjective, said detection path being configured to detect in said fieldof view, and according to a detection pattern, a light beam emitted bythe sample in response to said illumination of the sample, and togenerate a detection signal; a processing unit configured to generateinformation on microscopic analysis of the sample from said detectionsignal; a sighting path comprising: said microscope objective; afull-field illumination device configured to illuminate the sample in asecond spectral band; a two-dimensional detector; one or more imagingelements forming, with said microscope objective, a full-field imagingdevice configured to optically conjugate a given effective field of thesample encompassing said field of view with a detection area of thetwo-dimensional detector, and to form a sighting image in surfacereflection of said effective field; a beam splitter element arrangedupstream of the microscope objective in order to separate the analysispath and the sighting path; a display module configured to show saidsighting image and, on said sighting image, an image element indicatingthe position of said detection pattern.
 2. The system for analysis of asample as claimed in claim 1, in which the full-field imaging devicehas, in the object space of the microscope objective, a numericalaperture strictly less than the nominal numerical aperture of themicroscope objective.
 3. The system for analysis of a sample as claimedin claim 2, in which said sighting path further comprises a diaphragmfor limiting said numerical aperture of the full-field imaging device.4. The system for analysis of a sample as claimed in claim 1, in whichthe full-field imaging device of said sighting path is adjustable infocus.
 5. The system for analysis of a sample as claimed in claim 1, inwhich the full-field illumination device of the sighting path comprisesa plurality of light sources arranged on a periphery of a distal face ofthe microscope objective.
 6. The system for analysis of a sample asclaimed in claim 1, in which said second spectral band differs at leastpartially from said first spectral band, and said sighting pathcomprises means for reducing the light power at least in said firstspectral band.
 7. The system for analysis of a sample as claimed inclaim 1, in which said second spectral band differs at least partiallyfrom said first spectral band, and said beam splitter element comprisesa dichroic element configured to split the beams in each of said firstand second spectral bands.
 8. The system for analysis of a sample asclaimed in claim 1, in which said image element indicating the positionof said detection pattern comprises a graphic element determined bymeans of a prior calibration.
 9. The system for analysis of a sample asclaimed in claim 1, in which said microscopic analysis path is aconfocal imaging path and/or an optical coherence tomographic imagingpath, and said information on microscopic analysis of the samplecomprises at least one image of the sample.
 10. The system for analysisof a sample as claimed in claim 1, in which said microscopic analysispath is a spectroscopic analysis path, and said information onmicroscopic analysis of the sample comprises at least one spectrum ofsaid light beam emitted by the sample at at least one point of thesample.
 11. A method for analysis of a sample (S), comprising: amicroscopic analysis of the sample by means of a microscopic analysispath comprising a microscope objective of given nominal numericalaperture (NA) in a given field of view, said microscopic analysiscomprising: illuminating the sample through the microscope objectiveaccording to a first given illumination pattern and in a first spectralband; detecting in said field of view, and according to a detectionpattern, a light beam emitted by the sample in response to saidillumination of the sample in order to form a detection signal;processing said detection signal in order to generate information onmicroscopic analysis of the sample; the formation of a sighting image insurface reflection of a given effective field of the sample encompassingsaid field of view, by means of a sighting path comprising saidmicroscope objective, a two-dimensional detector, one or more imagingelements configured to form with said microscope objective a full-fieldimaging device, the formation of the sighting image comprising:full-field illumination of the sample in a second spectral band; opticalconjugation of the effective field of the sample with a detection areaof the two-dimensional detector, by means of said full-field imagingdevice, in order to form said sighting image; displaying said sightingimage and, on said sighting image, displaying an image elementindicating the position of said detection pattern.
 12. The method foranalysis of a sample as claimed in claim 11, in which the microscopicanalysis of the sample and the formation of a sighting image are carriedout continuously.
 13. The method for analysis of a sample as claimed inclaim 11, comprising: a first step of forming a sighting image of thesample without illumination of the microscopic analysis path, thedetection of an analysis zone of interest in the sighting image of thesample, and the microscopic analysis of said sample in said zone ofinterest.
 14. The method for analysis of a sample as claimed in claim13, in which the illumination of the sighting path is turned off duringthe microscopic analysis of the sample.
 15. The method for analysis of asample as claimed in claim 11, in which the microscopic analysis of thesample comprises confocal and/or optical coherence tomographic imagingof the sample.
 16. The method for analysis of a sample as claimed inclaim 15, in which the microscopic analysis of the sample comprises theformation of B-scan type images with a given imaging rate, and saidimaging rate is synchronized with a rate of acquisition of the sightingimages.
 17. The method for analysis of a sample as claimed in claim 11,in which the microscopic analysis of the sample comprises aspectroscopic analysis of the sample.
 18. The method for analysis of asample as claimed in claim 11, comprising a prior calibration stepmaking it possible to determine, for said image element, a graphicelement indicating the position of said detection pattern.
 19. Themethod for analysis of a sample as claimed in claim 11, furthercomprising displaying a marker superimposed on said image element of thesighting image, said marker allowing a user to target a point ofinterest in the detection pattern.
 20. The method for analysis of asample as claimed in claim 11, in which the sample is a biologicaltissue, for example skin.